Methods and systems for three-dimensional real-time intraoperative surgical margin evaluation of tumor tissues

ABSTRACT

A method of evaluating a surgical margin of tumor tissues of a living subject includes acquiring images of a specimen of the tumor tissues; calculating a three-dimensional (3D) morphological surface of the specimen from the acquired images and displaying the 3D morphological surface; obtaining, from the 3D morphological surface, a plurality of specimen locations to cover a surface of the specimen; acquiring optical data at each specimen location; evaluating a margin status of the specimen at each specimen location to either positive or negative based on the acquired optical data; and displaying the margin status of the specimen on the 3D morphological surface of the specimen with morphological orientations.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/085,732, filed Nov. 20, 2013, entitled “METHODS AND SYSTEMSFOR THREE-DIMENSIONAL REAL-TIME INTRAOPERATIVE SURGICAL MARGINEVALUATION OF TUMOR TISSUES,” by Anita Mahadevan-Jansen et al., nowallowed, which itself claims priority to and the benefit of, pursuant to35 U.S.C. §119(e), U.S. provisional patent application Ser. No.61/728,346, filed Nov. 20, 2012, entitled “METHODS AND SYSTEMS FOR 3D,REAL TIME AND INTRAOPERATIVE SURGICAL MARGIN EVALUATION,” by The QuyenNguyen et al., and U.S. provisional patent application Ser. No.61/821,442, field May 9, 2013, entitled “SYSTEMS AND METHODS FOR TUMORBED ASSESSMENT,” by The Quyen Nguyen et al., which are incorporatedherein in their entireties by reference.

Some references, which may include patents, patent applications andvarious publications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entiretiesand to the same extent as if each reference was individuallyincorporated by reference. In terms of notation, hereinafter, “[n]”represents the nth reference cited in the reference list. For example,[21] represents the 21st reference cited in the reference list, namely,Mahadevan-Jansen, A. in Raman Spectroscopy: From Benchtop to Bedside,Biomedical Photonics Handbook, (ed 30:1-30:27 T. Vo-Dinh, CRC Press,Washington D.C., 2003.).

STATEMENT AS TO RIGHTS UNDER FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under grant numberW81XWH 09-1-0037 awarded by the Department of Defense Breast CancerResearch Program. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to surgical procedures, and moreparticularly to methods and systems for evaluating surgical margins oftumor tissues of a living subject and a non-transitory computer-readablemedium storing instructions which, when executed by a processor, causethe system to perform the method for evaluating the surgical margin ofthe specimen.

BACKGROUND OF THE INVENTION

Soft tissue sarcomas (STS) are a heterogeneous group of malignant tumorsthat arise from mesenchymal tissues including fibrous connective tissue,cartilage, blood vessels, muscles, nerves, or fat. In the United States,it is estimated that some 11,280 new cases were diagnosed, and 3900patients died of STS in the year 2012 [1]. The mainstay of localtreatment is to completely excise the tumor with a margin of normaltissue so that no malignant cells remain in the tumor bed. The presenceof residual sarcoma cells in the tumor bed is associated with localrecurrence, which reduces patient survival rates [2-4]. For patientswith residual tumor cells at the margin of the resected tumor,re-excision and/or postoperative radiation is required. Such additionaltherapies increase patient morbidity and healthcare costs.

It has been shown that the presence of cancer cells within the margin ofresected specimens is strongly correlated with the risk of local tumorrecurrence. Margins therefore play a key role in the prognosis ofpatients with respect to local recurrence and are directly correlated tothe success of surgeries. Consequently, there is a need forintraoperative evaluation of the resection front so that immediatere-excision of suspicious margins can be performed.

Resected specimens differ in shape, size and firmness. Depending on thesize and stage of the tumor, the resected specimen can vary in shape andsize. The firmness of resected specimen is sometime related to age andbody mass index of the patient. This variation in size, sharp andfirmness makes the measurement of the specimen surface difficult.

In addition, there is no universal definition of a safe margin (i.e.,the thickness of healthy tissue surrounding the tumor). Depending on theorgan's location, the size of the safe surgical margin is defineddifferently. In fact, 2 mm is the safe margin widely accepted by breastsurgeons. But most urological surgeons will define the safe margin asthe absence of tumor at the surface of the removed prostate specimen.Accordingly, the safe margin should be no more than 0.05 mm in thiscase.

Generally, any method used to evaluate the surgical margins of aresected specimen must be precise, rapid and relatively simple toimplement in order to be used in routine clinical care. The methodshould be able to scan the entire surface of specimens despite theirdifferences in shape and size; and measure the sample surface whileleaving it intact, thus minimizing the physical change of the marginstatus due to pressure. Furthermore, the method should be able to beapplied for a wide range of surgery types for example breast, skin, andprostate cancer surgeries. Besides a precise diagnose of margin status,the method should also provide exact locations of the positive margin(if any are found) in a manner that the surgeon can easily recognize andcorrectly remove more tissues.

Therefore, a heretofore unaddressed need exists in the art to addressthe aforementioned deficiencies and inadequacies.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a method of evaluating asurgical margin of tumor tissues of a living subject, which includes:(a) acquiring images of a specimen of the tumor tissues; (b) calculatinga three-dimensional (3D) morphological surface of the specimen from theacquired images and displaying the 3D morphological surface; (c)obtaining, from the 3D morphological surface, a plurality of specimenlocations to cover a surface of the specimen; (d) acquiring optical dataat each specimen location; (e) evaluating a margin status of thespecimen at each specimen location to either positive or negative basedon the acquired optical data; and (f) displaying the margin status ofthe specimen on the 3D morphological surface of the specimen withmorphological orientations.

In certain embodiments, the step of acquiring the optical data includes:providing a source light; delivering the source light at each specimenlocation onto a surface of the specimen; and collectingdiffused/reflected light generated from interaction of the source lightwith the specimen at the specimen location.

In certain embodiments, the step of acquiring the optical data isperformed with at least one optical probe or at least one detector.

In certain embodiments, each optical probe includes a plurality ofoptical fibers spatially arranged in a fiber array. In certainembodiments, for each optical probe, the optical fibers include a sourcefiber for delivering the source light emitted from the light source tothe surface of the specimen, and a plurality of collection fibers forcollecting the diffused/reflected light generated from the interactionof the source light with the specimen. In certain embodiments, thesource fiber is positioned in a center of the fiber array, and theplurality of collection fibers is positioned in one or more rings havinga center at the source fiber such that each collection fiber is offsetfrom the source fiber.

In certain embodiments, the method further includes cooperatively movingthe specimen and the at least one optical probe to acquire the opticaldata at all of the specimen locations.

In certain embodiments, the method further includes: mounting thespecimen in a scanner; and calibrating a position of the specimen in thescanner. In certain embodiments, the scanner includes: a sample holderfor holding the specimen; a first motor for rotating the specimen alonga first horizontal axis; and a second motor for moving the at least oneoptical probe along a surface of the specimen. In certain embodiments,the scanner further includes a camera for acquiring images of thespecimen so as to reconstruct a three-dimensional (3D) morphologicalsurface of the specimen.

In certain embodiments, the evaluating step includes: generating afluorescence signal from the images acquired by the camera; anddetermining the margin status according to the fluorescence signal.

In certain embodiments, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a Raman spectrum (RS) fromthe raw spectrum; and determining the margin status according to the RS.

In certain embodiments, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a fluorescence spectrum (FS)and a Raman spectrum (RS) from the raw spectrum; and determining themargin status according to the FS and the RS.

In certain embodiments, the plurality of collection optical fibers isdivided into N groups, such that a first group of the collection opticalfibers each is positioned away along a radial direction from the sourceoptical fiber with a first spatial offset R₁, and a N-th group of thecollection optical fibers each is positioned away along the radialdirection from the source optical fiber with a N-th spatial offsetR_(N), where N is an positive integer, and R₁≦R₂≦ . . . ≦R_(N-1)≦R_(N).In one embodiment, the tumor tissues include breast cancer tissues, N=3,R₁=1.57 mm, R₂.=2.68 mm, and R₃=3.50 mm. In one embodiment, theevaluating step includes: determining the margin status using aspatially offset Raman spectroscopy (SORS) from the optical data.

In one embodiment, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a fluorescence spectrum (FS)from the raw spectrum; and determining the margin status according tothe FS.

In another aspect, the present invention relates to a system forevaluating a surgical margin of tumor tissues of a living subject. Inone embodiment, the system includes: (a) a light source configured toemit a source light; (b) at least one optical probe coupled with thelight source, each of the at least one optical probe has a working end,a source channel and a plurality of collection channels, wherein whenthe working end is positioned proximate to a surface of a specimen ofthe tumor tissues, the source channel is configured to deliver thesource light emitted by the light source from the working end to thesurface of the specimen, and the collection channels are configured tocollect from the working end diffused/reflected light generated frominteraction of the source light with the specimen; (c) a scanner coupledwith the at least one optical probe, configured to cooperatively movethe specimen and the at least one optical probe so as to probe aplurality of specimen locations, wherein the specimen locations coverthe surface of the specimen; (d) a detector coupled with the at leastone optical probe, configured to receive the collecteddiffused/reflected light to evaluate a margin status of the specimen;and (e) a controller coupled with the scanner and the detector,configured to operably control the scanner and the detector.

In certain embodiments, each optical probe includes a plurality ofoptical fibers spatially arranged in a fiber array. In certainembodiments, for each optical probe, the optical fibers include a sourcefiber for delivering the source light emitted from the light source tothe surface of the specimen, and a plurality of collection fibers forcollecting the diffused/reflected light generated from the interactionof the source light with the specimen. In certain embodiments, thesource fiber is positioned in a center of the fiber array, and theplurality of collection fibers is positioned in one or more rings havinga center at the source fiber such that each collection fiber is offsetfrom the source fiber.

In certain embodiments, the detector includes a spectrometer. In certainembodiments, the detector includes a charge-coupled device (CCD).

In certain embodiments, the detector is configured to: generate opticaldata from the received collected diffused/reflected light; obtain a rawspectrum from the optical data; generate a fluorescence spectrum (FS)and a Raman spectrum (RS) from the raw spectrum; and determine themargin status according to the FS and the RS.

In certain embodiments, the scanner includes: a sample holder forholding the specimen; a first motor for rotating the specimen along afirst horizontal axis; and a second motor for moving the at least oneoptical probe along a surface of the specimen. In certain embodiments,the scanner further includes a camera for acquiring images of thespecimen so as to reconstruct a three-dimensional (3D) morphologicalsurface of the specimen.

In certain embodiments, the detector is configured to: obtain a rawspectrum from the optical data; generate a Raman spectrum (RS) from theraw spectrum; and determine the margin status according to the RS.

In certain embodiments, the detector is configured to: generate afluorescence signal from the images acquired by the camera; anddetermine the margin status according to the fluorescence signal.

In certain embodiments, the detector is configured to: generate opticaldata from the received collected diffused/reflected light; obtain a rawspectrum from the optical data; generate a Raman spectrum (RS) from theraw spectrum; generate a fluorescence image (FI) from the imagesacquired by the camera; and determine the margin status according to theFI and the RS.

In certain embodiments, the controller includes a computer. In certainembodiments, the computer has a display for displaying the 3Dmorphological surface and displaying the margin status of the specimenin the 3D morphological surface with morphological orientations.

In certain embodiments, the plurality of collection optical fibers isdivided into N groups, such that a first group of the collection opticalfibers each is positioned away along a radial direction from the sourceoptical fiber with a first spatial offset R₁, and a N-th group of thecollection optical fibers each is positioned away along the radialdirection from the source optical fiber with a N-th spatial offsetR_(N), where N is an positive integer, and R₁≦R₂≦ . . . ≦R_(N-1)≦R_(N).In one embodiment, the tumor tissues include breast cancer tissues, N=3,R₁=1.57 mm, R₂.=2.68 mm, and R₃=3.50 mm. In one embodiment, the detectoris configured to: generate the optical data from the collecteddiffused/reflected light; obtain a raw spectrum from the optical datacollected by each of the N groups of the collection optical fibers;generate a Raman spectrum (RS) from the raw spectrum collected by eachof the N groups of the collection optical fiber; and determine themargin status according to the RS collected by each of the N groups ofthe collection optical fiber.

In one embodiment, the detector is configured to: generate optical datafrom the received collected diffused/reflected light; obtain a rawspectrum from the optical data; generate a fluorescence spectrum (FS)from the raw spectrum; and determine the margin status according to theFS.

A further aspect of the present invention relates to a non-transitorycomputer-readable medium storing computer executable instructions which,when executed by a processor, cause a system to perform a method forevaluating a surgical margin of tumor tissues of a living subject. Incertain embodiments, the method includes: (a) acquiring images of aspecimen of the tumor tissues; (b) calculating a three-dimensional (3D)morphological surface of the specimen from the acquired images anddisplaying the 3D morphological surface; (c) obtaining, from the 3Dmorphological surface, a plurality of specimen locations to cover asurface of the specimen; (d) acquiring optical data at each specimenlocation; (e) evaluating a margin status of the specimen at eachspecimen location to either positive or negative based on the acquiredoptical data; and (f) displaying the margin status of the specimen onthe 3D morphological surface of the specimen with morphologicalorientations.

In certain embodiments, the step of acquiring the optical data includes:providing a source light; delivering the source light at each specimenlocation onto the surface of the specimen; collecting diffused/reflectedlight generated from interaction of the source light with the specimenat the specimen location; and evaluate the margin status of the specimenfrom the collected diffused/reflected light at each specimen location ofthe surface of the specimen.

In certain embodiments, the step of acquiring the optical data isperformed with at least one optical probe or at least one detector.

In certain embodiments, the method further includes cooperatively movingthe specimen and the at least one optical probe to acquire the opticaldata at all of the specimen locations.

In certain embodiments, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a fluorescence spectrum (FS)and a Raman spectrum (RS) from the raw spectrum; and determining themargin status according to the FS and the RS.

In certain embodiments, the method further includes: mounting thespecimen in a scanner; and calibrating a position of the specimen in thescanner. In certain embodiments, the scanner includes a camera foracquiring images of the specimen so as to reconstruct athree-dimensional (3D) morphological surface of the specimen.

In certain embodiments, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a Raman spectrum (RS) fromthe raw spectrum; and determining the margin status according to the RS.

In certain embodiments, the evaluating step includes: generating afluorescence signal from the images acquired by the camera; anddetermining the margin status according to the fluorescence signal.

In certain embodiments, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a Raman spectrum (RS) fromthe raw spectrum; generating a fluorescence image (FI) from the imagesacquired by the camera; and determining the margin status according tothe FI and the RS.

In certain embodiments, the at least one optical probe includes: (a) asource optical fiber; and (b) a plurality of collection optical fibersarranged in an array, wherein the plurality of collection optical fibersis divided into N groups, such that a first group of the collectionoptical fibers each is positioned away along a radial direction from thesource optical fiber with a first spatial offset R₁, and a N-th group ofthe collection optical fibers each is positioned away along the radialdirection from the source optical fiber with a N-th spatial offsetR_(N), where N is an positive integer, and R₁≦R₂≦ . . . ≦R_(N-1)≦R_(N).In one embodiment, the tumor tissues include breast cancer tissues, N=3,R₁=1.57 mm, R₂.=2.68 mm, and R₃=3.50 mm. In one embodiment, theevaluating step includes: generating the optical data from the collecteddiffused/reflected light; obtaining a raw spectrum from the optical datacollected by each of the N groups of the collection optical fibers;generating a Raman spectrum (RS) from the raw spectrum collected by eachof the N groups of the collection optical fiber; and determining themargin status according to the RS collected by each of the N groups ofthe collection optical fiber.

In one embodiment, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a fluorescence spectrum (FS)from the raw spectrum; and determining the margin status according tothe FS.

In another aspect, the present invention relates to a method ofevaluating a surgical margin of tumor tissues of a living subject. Inone embodiment, the method includes: (a) reconstructing athree-dimensional (3D) morphological surface of a specimen of the tumortissues; (b) automatically acquiring optical data at a plurality ofspecimen locations on a surface of the specimen, wherein the specimenlocations cover a surface of the specimen; (c) automatically evaluatinga margin status of the specimen based on the acquired optical data ateach specimen location on the surface of the specimen; and (d)displaying the margin status of the specimen on the 3D morphologicalsurface of the specimen with morphological orientations.

In certain embodiments, the reconstructing step includes: acquiringimages of the specimen; and reconstructing the three-dimensional (3D)morphological surface of the specimen based on the acquired images ofthe specimen.

In certain embodiments, the acquiring step includes: providing a sourcelight; delivering the source light at each specimen location onto thesurface of the specimen; and collecting diffused/reflected lightgenerated from interaction of the source light with the specimen at thespecimen location.

In certain embodiments, the acquiring step is performed with at leastone optical probe or at least one detector, and wherein each opticalprobe comprises a plurality of optical fibers spatially arranged in afiber array. In certain embodiments, for each optical probe, the opticalfibers include a source fiber for delivering the source light emittedfrom the light source to the surface of the specimen, and a plurality ofcollection fibers for collecting the diffused/reflected light generatedfrom the interaction of the source light with the specimen. In certainembodiments, the source fiber is positioned in a center of the fiberarray, and the plurality of collection fibers is positioned in one ormore rings having a center at the source fiber such that each collectionfiber is offset from the source fiber.

In certain embodiments, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a fluorescence spectrum (FS)and a Raman spectrum (RS) from the raw spectrum; and determining themargin status according to the FS and the RS.

In certain embodiments, the method further includes: calculating thespecimen locations to cover the surface of the specimen.

In certain embodiments, the method further includes: mounting thespecimen in a scanner; and calibrating a position of the specimen in thescanner. In certain embodiments, the scanner includes: a sample holderfor holding the specimen; a first motor for rotating the specimen alonga first horizontal axis; and a second motor for moving the at least oneoptical probe along a surface of the specimen. In certain embodiments,the scanner further includes a camera for acquiring images of thespecimen so as to reconstruct a three-dimensional (3D) morphologicalsurface of the specimen.

In certain embodiments, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a Raman spectrum (RS) fromthe raw spectrum; and determining the margin status according to the RS.

In certain embodiments, the evaluating step includes: generating afluorescence signal from the images acquired by the camera; anddetermining the margin status according to the fluorescence signal.

In certain embodiments, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a Raman spectrum (RS) fromthe raw spectrum; generating a fluorescence image (FI) from the imagesacquired by the camera; and determining the margin status according tothe FI and the RS.

In certain embodiments, the plurality of collection optical fibers isdivided into N groups, such that a first group of the collection opticalfibers each is positioned away along a radial direction from the sourceoptical fiber with a first spatial offset R₁, and a N-th group of thecollection optical fibers each is positioned away along the radialdirection from the source optical fiber with a N-th spatial offsetR_(N), where N is an positive integer, and R₁≦R₂≦ . . . ≦R_(N-1)≦R_(N).In one embodiment, the tumor tissues include breast cancer tissues, N=3,R₁=1.57 mm, R₂.=2.68 mm, and R₃=3.50 mm. In one embodiment, theevaluating step includes: generating the optical data from the collecteddiffused/reflected light; obtaining a raw spectrum from the optical datacollected by each of the N groups of the collection optical fibers;generating a Raman spectrum (RS) from the raw spectrum collected by eachof the N groups of the collection optical fiber; and determining themargin status according to the RS collected by each of the N groups ofthe collection optical fiber.

In one embodiment, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a fluorescence spectrum (FS)from the raw spectrum; and determining the margin status according tothe FS.

In yet another aspect, a system for evaluating a surgical margin oftumor tissues of a living subject includes: (a) a reconstruction deviceconfigured to reconstruct a three-dimensional (3D) morphological surfaceof a specimen of the tumor tissues; (b) an acquiring device configuredto acquiring optical data at a plurality of specimen locations, whereinthe specimen locations cover a surface of the specimen; (c) anevaluating device configured to evaluate a margin status of the specimenbased on the acquired optical data at each specimen location on thesurface of the specimen; (d) a display device configured to display the3D morphological surface and to display the margin status of thespecimen on the 3D morphological surface of the specimen withmorphological orientations.

In certain embodiments, the acquiring device includes: a light sourceconfigured to emit a source light; at least one optical probe coupledwith the light source, each of the at least one optical probe has aworking end, a source channel and a plurality of collection channels,wherein when the working end is positioned proximate to a surface of aspecimen of the tumor tissues, the source channel is configured todeliver the source light emitted by the light source from the workingend to the surface of the specimen, and the collection channels areconfigured to collect from the working end diffused/reflected lightgenerated from interaction of the source light with the specimen; and ascanner coupled with the at least one optical probe, configured tocooperatively move the specimen and the at least one optical probe so asto probe each of the specimen locations.

In certain embodiments, each optical probe includes a plurality ofoptical fibers spatially arranged in a fiber array. In certainembodiments, for each optical probe, the optical fibers include a sourcefiber for delivering the source light emitted from the light source tothe surface of the specimen, and a plurality of collection fibers forcollecting the diffused/reflected light generated from the interactionof the source light with the specimen. In certain embodiments, thesource fiber is positioned in a center of the fiber array, and theplurality of collection fibers is positioned in one or more rings havinga center at the source fiber such that each collection fiber is offsetfrom the source fiber.

In certain embodiments, the scanner includes: a sample holder forholding the specimen; a first motor for rotating the specimen along afirst horizontal axis; and a second motor for moving the at least oneoptical probe along the surface of the specimen.

In certain embodiments, the evaluating device includes a detectorcoupled to the at least one optical probe, configured to receive thecollected diffused/reflected light to evaluate the margin status of thespecimen.

In certain embodiments, the detector is configured to: generate opticaldata from the received collected diffused/reflected light; obtain a rawspectrum from the optical data; generate a fluorescence spectrum (FS)and a Raman spectrum (RS) from the raw spectrum; and determine themargin status according to the FS and the RS.

In certain embodiments, the reconstruction device includes: a camera foracquiring images of the specimen; and a processor for reconstructing the3D morphological surface of the specimen based on the acquired images ofthe specimen.

In certain embodiments, the detector is configured to: obtain a rawspectrum from the optical data; generate a Raman spectrum (RS) from theraw spectrum; and determine the margin status according to the RS.

In certain embodiments, the detector is configured to: generate afluorescence signal from the images acquired by the camera; anddetermine the margin status according to the fluorescence signal.

In certain embodiments, the detector is configured to: generate opticaldata from the received collected diffused/reflected light; obtain a rawspectrum from the optical data; generate a Raman spectrum (RS) from theraw spectrum; generate a fluorescence image (FI) from the imagesacquired by the camera; and determine the margin status according to theFI and the RS.

In certain embodiments, the plurality of collection optical fibers isdivided into N groups, such that a first group of the collection opticalfibers each is positioned away along a radial direction from the sourceoptical fiber with a first spatial offset R₁, and a N-th group of thecollection optical fibers each is positioned away along the radialdirection from the source optical fiber with a N-th spatial offsetR_(N), where N is an positive integer, and R₁≦R₂≦ . . . ≦R_(N-1)≦R_(N).In one embodiment, the tumor tissues include breast cancer tissues, N=3,R₁=1.57 mm, R₂.=2.68 mm, and R₃=3.50 mm. In one embodiment, the detectoris configured to: generate the optical data from the collecteddiffused/reflected light; obtain a raw spectrum from the optical datacollected by each of the N groups of the collection optical fibers;generate a Raman spectrum (RS) from the raw spectrum collected by eachof the N groups of the collection optical fiber; and determine themargin status according to the RS collected by each of the N groups ofthe collection optical fiber.

In one embodiment, the detector is configured to: generate optical datafrom the received collected diffused/reflected light; obtain a rawspectrum from the optical data; generate a fluorescence spectrum (FS)from the raw spectrum; and determine the margin status according to theFS.

In a further aspect, the present invention relates to a non-transitorycomputer-readable medium storing instructions which, when executed by aprocessor, cause a system to perform a method of evaluating a surgicalmargin of tumor tissues of a living subject. In one embodiment, themethod includes: (a) reconstructing a three-dimensional (3D)morphological surface of a specimen of the tumor tissues; (b)automatically acquiring optical data at a plurality of specimenlocations on a surface of the specimen, wherein the specimen locationscover a surface of the specimen; (c) automatically evaluating a marginstatus of the specimen based on the acquired optical data at eachspecimen location on the surface of the specimen; and (d) displaying themargin status of the specimen on the 3D morphological surface of thespecimen with morphological orientations.

In certain embodiments, the reconstructing step includes: acquiringimages of the specimen; and reconstructing the three-dimensional (3D)morphological surface of the specimen based on the acquired images ofthe specimen.

In certain embodiments, the acquiring step includes: providing a sourcelight; delivering the source light at each specimen location onto thesurface of the specimen; and collecting diffused/reflected lightgenerated from interaction of the source light with the specimen at thespecimen location.

In certain embodiments, the acquiring step is performed with at leastone optical probe or at least one detector, and each optical probeincludes a plurality of optical fibers spatially arranged in a fiberarray. In certain embodiments, for each optical probe, the opticalfibers include a source fiber for delivering the source light emittedfrom the light source to the surface of the specimen, and a plurality ofcollection fibers for collecting the diffused/reflected light generatedfrom the interaction of the source light with the specimen. In certainembodiments, the source fiber is positioned in a center of the fiberarray, and the plurality of collection fibers is positioned in one ormore rings having a center at the source fiber such that each collectionfiber is offset from the source fiber.

In certain embodiments, the method further includes cooperatively movingthe specimen and the at least one optical probe to acquire the opticaldata at all of the specimen locations.

In certain embodiments, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a fluorescence spectrum (FS)and a Raman spectrum (RS) from the raw spectrum; and determining themargin status according to the FS and the RS.

In certain embodiments, the method further includes: calculating thespecimen locations to cover the surface of the specimen.

In certain embodiments, the method further includes: mounting thespecimen in a scanner; and calibrating a position of the specimen in thescanner. In certain embodiments, the scanner includes a camera foracquiring images of the specimen so as to reconstruct athree-dimensional (3D) morphological surface of the specimen.

In certain embodiments, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a Raman spectrum (RS) fromthe raw spectrum; and determining the margin status according to the RS.

In certain embodiments, the evaluating step includes: generating afluorescence signal from the images acquired by the camera; anddetermining the margin status according to the fluorescence signal.

In certain embodiments, the evaluating step includes: obtaining a rawspectrum from the optical data; generating a Raman spectrum (RS) fromthe raw spectrum; generating a fluorescence image (FI) from the imagesacquired by the camera; and determining the margin status according tothe FI and the RS.

In a further aspect, the present invention relates to an optical probefor evaluating boundary of tumor tissues of a living subject. In oneembodiment, the optical probe includes: (a) a source optical fiber; and(b) a plurality of collection optical fibers arranged in an array,wherein the plurality of collection optical fibers is divided into Ngroups, such that a first group of the collection optical fibers each ispositioned away along a radial direction from the source optical fiberwith a first spatial offset R₁, and a N-th group of the collectionoptical fibers each is positioned away along the radial direction fromthe source optical fiber with a N-th spatial offset R_(N), where N is anpositive integer, and R₁≦R₂≦ . . . ≦R_(N-1)≦R_(N).

In certain embodiments, the source optical fiber is positioned in acenter of the optical probe, and each of the N groups of the collectionoptical fibers is positioned in a ring having a center at the sourceoptical fiber such that each of the collection optical fibers in theN-th group is offset from the source optical fiber with the N-th spatialoffset R_(N). In one embodiment, the tumor tissues include breast cancertissues, N=3, R₁=1.57 mm, R₂.=2.68 mm, and R₃=3.50 mm.

These and other aspects of the present invention will become apparentfrom the following description of the preferred embodiment taken inconjunction with the following drawings, although variations andmodifications therein may be affected without departing from the spiritand scope of the novel concepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of theinvention and together with the written description, serve to explainthe principles of the invention. Wherever possible, the same referencenumbers are used throughout the drawings to refer to the same or likeelements of an embodiment.

FIG. 1 shows a typical tumor bed after the STS excision according tocertain embodiments of the present invention.

FIG. 2A shows schematically a system for evaluating a surgical margin oftumor tissues of a living subject according to certain embodiments ofthe present invention.

FIG. 2B shows schematically a scanner utilized in the system as shown inFIG. 2A according to certain embodiments of the present invention.

FIG. 2C shows schematically a system for evaluating a surgical margin oftumor tissues of a living subject according to certain embodiments ofthe present invention.

FIGS. 3A and 3B show schematically two views of a system for evaluatinga surgical margin of a specimen of a living subject according to certainembodiments of the present invention.

FIG. 4 shows schematically (A) a multi-fiber probe according to certainembodiments of the invention, (B) multi-fibers aligned at the entranceof the detector according to certain embodiments of the invention, and(C) a zoom view of the working end of the probe according to certainembodiments of the invention.

FIG. 5 shows the source-detector (S-D) offset for an equivalent S/N ofthe optical probe according to certain embodiments of the presentinvention.

FIG. 6 shows schematically (A) a breast resected specimen according tocertain embodiments of the present invention, and (B) tumor positivemargins and negative margins according to certain embodiments of thepresent invention.

FIG. 7 shows the margin status of a phantom sample that mimics anexcised cancer tumor according to certain embodiments of the presentinvention.

FIG. 8 shows the measured spectrums of a tissue sample according tocertain embodiments of the present invention, wherein (A) shows a rawspectrum, and (B) shows a fluorescence spectrum (FS) and a Ramanspectrum (RS) obtained from the raw spectrum of (A).

FIG. 9 shows a flowchart of a method of evaluating a surgical margin ofa specimen of a living subject according to certain embodiments of thepresent invention.

FIG. 10A shows schematically average Raman spectra of control muscle andSTS samples according to certain embodiments of the present invention.

FIG. 10B shows schematically mean Raman spectra and associatedbiochemical peaks according to certain embodiments of the presentinvention.

FIG. 11 shows (a) fluorescence spectra of muscle and STS, (b) an imageof the surface of a samples and the boundary of STS and normal muscle,and (c) fluorescence intensity (mean±standard deviation) of the tworegions according to certain embodiments of the present invention.

FIG. 12 shows the probability of individual samples being either normalmuscle or tumor muscle according to certain embodiments of the presentinvention.

FIG. 13 shows schematically Raman spectra of in vivo tissues accordingto certain embodiments of the present invention.

FIG. 14 shows Fluorescence intensity (mean±standard deviation) of muscleand tumor from 19 patients according to certain embodiments of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is more particularly described in the followingexamples that are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art. Various embodiments of the invention are now described indetail. Referring to the drawings, like numbers indicate like componentsthroughout the views. As used in the description herein and throughoutthe claims that follow, the meaning of “a”, “an”, and “the” includesplural reference unless the context clearly dictates otherwise. Also, asused in the description herein and throughout the claims that follow,the meaning of “in” includes “in” and “on” unless the context clearlydictates otherwise. Moreover, titles or subtitles may be used in thespecification for the convenience of a reader, which shall have noinfluence on the scope of the present invention. Additionally, someterms used in this specification are more specifically defined below.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. For convenience, certainterms may be highlighted, for example using italics and/or quotationmarks. The use of highlighting has no influence on the scope and meaningof a term; the scope and meaning of a term is the same, in the samecontext, whether or not it is highlighted. It will be appreciated thatsame thing can be said in more than one way. Consequently, alternativelanguage and synonyms may be used for any one or more of the termsdiscussed herein, nor is any special significance to be placed uponwhether or not a term is elaborated or discussed herein. Synonyms forcertain terms are provided. A recital of one or more synonyms does notexclude the use of other synonyms. The use of examples anywhere in thisspecification including examples of any terms discussed herein isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to various embodiments given in this specification.

It will be understood that when an element is referred to as being “on”another element, it can be directly on the other element or interveningelements may be present therebetween. In contrast, when an element isreferred to as being “directly on” another element, there are nointervening elements present. As used herein, the term “and/or” includesany and all combinations of one or more of the associated listed items.

It will be understood that, although the terms first, second, third etc.may be used herein to describe various elements, components, regions,layers and/or sections, these elements, components, regions, layersand/or sections should not be limited by these terms. These terms areonly used to distinguish one element, component, region, layer orsection from another element, component, region, layer or section. Thus,a first element, component, region, layer or section discussed belowcould be termed a second element, component, region, layer or sectionwithout departing from the teachings of the present invention.

Furthermore, relative terms, such as “lower” or “bottom” and “upper” or“top,” may be used herein to describe one element's relationship toanother element as illustrated in the Figures. It will be understoodthat relative terms are intended to encompass different orientations ofthe device in addition to the orientation depicted in the Figures. Forexample, if the device in one of the figures is turned over, elementsdescribed as being on the “lower” side of other elements would then beoriented on “upper” sides of the other elements. The exemplary term“lower”, can therefore, encompasses both an orientation of “lower” and“upper,” depending of the particular orientation of the figure.Similarly, if the device in one of the figures is turned over, elementsdescribed as “below” or “beneath” other elements would then be oriented“above” the other elements. The exemplary terms “below” or “beneath”can, therefore, encompass both an orientation of above and below.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art and thepresent disclosure, and will not be interpreted in an idealized oroverly formal sense unless expressly so defined herein.

As used herein, “around”, “about” or “approximately” shall generallymean within 20 percent, preferably within 10 percent, and morepreferably within 5 percent of a given value or range. Numericalquantities given herein are approximate, meaning that the term “around”,“about” or “approximately” can be inferred if not expressly stated.

As used herein, “plurality” means two or more.

As used herein, the terms “comprising”, “including”, “carrying”,“having”, “containing”, “involving”, and the like are to be understoodto be open-ended, i.e., to mean including but not limited to.

The term “tissues” or “tumor tissues,” as used herein, refers to acollection of cells of a living subject (e.g., a human being or otheranimals). In certain embodiments, the tissues may include tissues in oron the body of the living subject, and/or tissues resected from the bodyof the living subject.

The term “specimen” of the tissues, as used herein, refers to samples ofbody tissues. The specimen of the tissues may be scanned or processed onsite (i.e., in or on the body of the living subject), or may be resectedfrom the body for scanning and processing purposes.

Overview of the Invention

The present invention relates to methods and systems for evaluatingsurgical margins of tumor tissues of a living subject and anon-transitory computer-readable medium storing instructions which, whenexecuted by a processor, cause the system to perform the method forevaluating the surgical margin of the specimen.

Resected specimens differ in shape, size and firmness. Depending on thesize and stage of the tumor, the resected specimen can vary in shape andsize. The firmness of resected specimen is sometime related to age andbody mass index of the patient. This variation in size, sharp andfirmness makes the measurement of the specimen surface difficult.

In addition, there is no universal definition of a safe margin (i.e.,the thickness of healthy tissue surrounding the tumor). Depending on theorgan's location, the size of the safe surgical margin is defineddifferently. In fact, 2 mm is the safe margin widely accepted by breastsurgeons. But most urological surgeons will define the safe margin asthe absence of tumor at the surface of the removed prostate specimen.Accordingly, the safe margin should be no more than 0.05 mm in thiscase.

Generally, any method used to evaluate the surgical margins of aresected specimen must be precise, rapid and relatively simple toimplement in order to be used in routine clinical care. The methodshould be able to scan the entire surface of specimens despite theirdifferences in shape and size; and measure the sample surface whileleaving it intact, thus minimizing the physical change of the marginstatus due to pressure. Furthermore, the method should be able to beapplied for a wide range of surgery types for example breast, skin, andprostate cancer surgeries. Besides a precise diagnose of margin status,the method should also provide exact locations of the positive margin(if any are found) in a manner that the surgeon can easily recognize andcorrectly remove more tissues.

As described above, a method used to evaluate the surgical margins of aresected specimen has the follow three requirements:

(a) Precise diagnose of margin status: the method should have a highsensitivity and specificity in evaluating the margin. The device shouldmeasure the sample surface while leaving it intact, thus minimizing thephysical change of the margin status due to pressure, which will limitthe false positive diagnosis.

(b) Rapid diagnosis and simple to implement in routine clinical care:the method should perform automatic measurement, without humanintervention, and the total measurement time must be less than 20minutes so that the measurement does not interfere with the surgeryplan. The device should be able to automatically scan the entire surfaceof the sample and provide exact locations of the positive margin (if anyare found) in a manner that the surgeon can easily recognize andcorrectly remove more tissues.

(c) Different margin size configuration: the margin size can vary fromseveral microns to several millimeters for different margin sizeapplications.

Current intraoperative margin assessment methods have limitations.Frozen sections are commonly used but are time consuming and prone tosampling error [5] especially with large tumor beds. For example, FIG. 1shows a typical tumor bed after the STS excision according to certainembodiments of the present invention. As shown in FIG. 1, the rectanglesmarked the biopsied locations for frozen section pathology. Magneticresonance imaging (MRI) provides general evaluation of the extent oftumor and thus allows for image-guided surgery, but has limitedsensitivity [6]. Serial sectioning of the resected tumor with standardhistopathology provides a definitive diagnosis of margin status, butresults may take from several days to over a week.

Limitations in current methods therefore highlight the need for a rapid,accurate, automated guidance tool that can be used in the operating roomduring tumor resection so that immediate re-excision of suspiciousmargins can be performed, minimizing the necessity for a second surgeryand its associated risks. This would significantly improve themanagement of the STS disease regarding both time and cost. It will cost$6,000-$10,000 for re-excision surgery and postoperative radiation;those additional procedures, following incomplete tumor removal,increase morbidity rates and cause significant physical, emotional,mental, and economic stress for patients.

In the present disclosure, we propose a solution using source light toevaluate the surgical margin. In certain embodiments, the systemutilizes optical devices to detect the presence of any microscopicresidual sarcoma cells in the tumor bed, report their locations tosurgeons so that more tissue can be removed, and thus help to ensurecomplete removal of the tumor with clear margins in a single procedure.The system would solve a critical problem in STS care by greatlyreducing or eliminating the need for re-operation/radiation as well asreducing the time, cost, and anxiety associated with repeat surgeriesand additional radiations

One aspect of the present invention relates to a method of evaluating asurgical margin of tumor tissues of a living subject, which includes:(a) acquiring images of a specimen of the tumor tissues; (b) calculatinga three-dimensional (3D) morphological surface of the specimen from theacquired images and displaying the 3D morphological surface; (c)obtaining, from the 3D morphological surface, a plurality of specimenlocations to cover a surface of the specimen; (d) acquiring optical dataat each specimen location; (e) evaluating a margin status of thespecimen at each specimen location to either positive or negative basedon the acquired optical data; and (f) displaying the margin status ofthe specimen on the 3D morphological surface of the specimen withmorphological orientations.

In another aspect, the present invention relates to a system forevaluating a surgical margin of tumor tissues of a living subject. Inone embodiment, the system includes: (a) a light source configured toemit a source light; (b) at least one optical probe coupled with thelight source, each of the at least one optical probe has a working end,a source channel and a plurality of collection channels, wherein whenthe working end is positioned proximate to a surface of a specimen ofthe tumor tissues, the source channel is configured to deliver thesource light emitted by the light source from the working end to thesurface of the specimen, and the collection channels are configured tocollect from the working end diffused/reflected light generated frominteraction of the source light with the specimen; (c) a scanner coupledwith the at least one optical probe, configured to cooperatively movethe specimen and the at least one optical probe so as to probe aplurality of specimen locations, wherein the specimen locations coverthe surface of the specimen; (d) a detector coupled with the at leastone optical probe, configured to receive the collecteddiffused/reflected light to evaluate a margin status of the specimen;and (e) a controller coupled with the scanner and the detector,configured to operably control the scanner and the detector.

A further aspect of the present invention relates to a non-transitorycomputer-readable medium storing computer executable instructions which,when executed by a processor, cause a system to perform a method forevaluating a surgical margin of tumor tissues of a living subject. Incertain embodiments, the method includes: (a) acquiring images of aspecimen of the tumor tissues; (b) calculating a three-dimensional (3D)morphological surface of the specimen from the acquired images anddisplaying the 3D morphological surface; (c) obtaining, from the 3Dmorphological surface, a plurality of specimen locations to cover asurface of the specimen; (d) acquiring optical data at each specimenlocation; (e) evaluating a margin status of the specimen at eachspecimen location to either positive or negative based on the acquiredoptical data; and (f) displaying the margin status of the specimen onthe 3D morphological surface of the specimen with morphologicalorientations.

In another aspect, the present invention relates to a method ofevaluating a surgical margin of tumor tissues of a living subject. Inone embodiment, the method includes: (a) reconstructing athree-dimensional (3D) morphological surface of a specimen of the tumortissues; (b) automatically acquiring optical data at a plurality ofspecimen locations on a surface of the specimen, wherein the specimenlocations cover a surface of the specimen; (c) automatically evaluatinga margin status of the specimen based on the acquired optical data ateach specimen location on the surface of the specimen; and (d)displaying the margin status of the specimen on the 3D morphologicalsurface of the specimen with morphological orientations.

In yet another aspect, a system for evaluating a surgical margin oftumor tissues of a living subject includes: (a) a reconstruction deviceconfigured to reconstruct a three-dimensional (3D) morphological surfaceof a specimen of the tumor tissues; (b) an acquiring device configuredto acquiring optical data at a plurality of specimen locations, whereinthe specimen locations cover a surface of the specimen; (c) anevaluating device configured to evaluate a margin status of the specimenbased on the acquired optical data at each specimen location on thesurface of the specimen; (d) a display device configured to display the3D morphological surface and to display the margin status of thespecimen on the 3D morphological surface of the specimen withmorphological orientations.

In a further aspect, the present invention relates to a non-transitorycomputer-readable medium storing instructions which, when executed by aprocessor, cause a system to perform a method of evaluating a surgicalmargin of tumor tissues of a living subject. In one embodiment, themethod includes: (a) reconstructing a three-dimensional (3D)morphological surface of a specimen of the tumor tissues; (b)automatically acquiring optical data at a plurality of specimenlocations on a surface of the specimen, wherein the specimen locationscover a surface of the specimen; (c) automatically evaluating a marginstatus of the specimen based on the acquired optical data at eachspecimen location on the surface of the specimen; and (d) displaying themargin status of the specimen on the 3D morphological surface of thespecimen with morphological orientations.

In certain embodiments, the non-transitory tangible computer-readablestorage medium includes, but not limited to, disk, CD-ROM, read-onlymemory (ROM), random memory (RAM), flash dive, or the likes.

These and other aspects of the present invention are more specificallydescribed below.

Implementations and Examples of the Invention

Without intent to limit the scope of the invention, exemplary methodsand their related results according to the embodiments of the presentinvention are given below. Note that titles or subtitles may be used inthe examples for convenience of a reader, which in no way should limitthe scope of the invention. Moreover, certain theories are proposed anddisclosed herein; however, in no way they, whether they are right orwrong, should limit the scope of the invention so long as the inventionis practiced according to the invention without regard for anyparticular theory or scheme of action.

One aspect of the present invention relates to a system for evaluating asurgical margin of tumor tissues of a living subject. In certainembodiments, the system is capable of measuring the entire surface of aspecimen in real time, and can perform at least the following tasks:

(1) reconstructing the 3D morphology of the specimen;

(2) automatically measuring the entire surface of the specimen;

(3) automatically evaluating the surgical margin status based on thepresence of a cancer signal in optical signal recorded at each location;and

(4) displaying the surgical margin status in 3D with morphologicalorientations of the specimen.

Further, the system provides new solutions and fulfills the threerequirements:

Precise: The system measures the margin status while leaving the sampleintact. Very little/no pressure will be put on the sample surface. Therisk of false positive margin due to surface deformation is thusminimized. Combining with high sensitivity and specificity in the marginevaluation of the optical technique, the system thus gives rise toprecise diagnosis of the margin status.

Rapid and simple: As the 3D morphology of the sample is known, theentire surface of the sample can be automatically scanned. The marginstatus of the entire specimen can be automatically evaluated withouthuman intervention.

Various margin sizes: As the system can be used with different opticaltechniques, it can be used in different margin size settings. In fact,different optical techniques have different depth resolutions which canrange from several microns to several millimeters. By selecting anappropriate optical technique, one can obtain an appropriate depthresolution for a specific margin application.

FIG. 2A shows schematically a system for evaluating a surgical margin oftumor tissues of a living subject according to certain embodiments ofthe present invention. Referring to FIG. 2A, the system 100 includes alight source 110, a spectrometer 120 with a detector, for example, acharge-coupled device (CCD) 125, multiples optical probes 130 and ascanner 140. A computer 150 is used to control the scanner 140 and thespectrometer 120.

The light source 110 is configured to emit a source light. The opticalprobes 130 are coupled with the light source 110. Each of the at leastone optical probe has a working end, a source channel and a plurality ofcollection channels, which will be described later.

The computer 150 serves as a controller of the system 100. In certainembodiments, the computer 150 may include a display for displaying the3D morphological surface and displaying the margin status of thespecimen in the 3D morphological surface with morphologicalorientations.

FIG. 2B shows schematically a scanner utilized in the system as shown inFIG. 2A according to certain embodiments of the present invention. Asshown in FIG. 2B, the scanner 140 includes two motors 141 and 142, aprobe holder (143, 144), and a camera 145. The probe holder has a firstmember 143 having a first end portion 143 a and an opposite, second endportion 143 b, and a second member 144 having a first end portion 144 aand an opposite, second end portion 144 b. The first and second endportions 144 a and 144 b of the second member 144 are respectivelyconnected to the at least one optical probe 130 (e.g., four opticalprobes shown in FIG. 2B according to one embodiment of the invention)and the first end portion 143 a of the first member 143 such that thefirst member 143 and the second member 144 are perpendicular to eachother. The use of four optical probes 130 mounted on the probe holder ofthe scanner 140 is demonstrated. The optical probes 130 have a workingend (i.e., the bottom end) placed proximal to the surface 102 of thespecimen 101. The first motor 141 operably rotates the specimen 101around its horizontal axis 148. The second motor 142 has an output shaft142 a extending along a second horizontal axis 149 that has an anglerelative to the first horizontal axis 148. The angle between the firstand second horizontal axes 148 and 149 is greater than zero. In theexemplary embodiment shown in FIG. 2B, the first and second horizontalaxes 148 and 149 is substantially perpendicular to each other. Theoutput shaft 142 a is connected to the second end portion 143 b of thefirst member 143 of the probe holder such that the output shaft 142 a isperpendicular to the first member 143. The output shaft 142 a of thesecond motor 142 operably rotates around the second horizontal axis 149,rotation of the output shaft 142 a of the second motor 142 drives thefirst and second members 143 and 144 of the probe holder to rotatearound the second horizontal axis 149, which in turn drives the at leastone optical probe 130 to rotate around the second horizontal axis 149.As such arrangement, in operation, the first motor 141 rotates thespecimen 101 around the first horizontal axis 148, while the secondmotor 142 rotates the optical probes 130 over the surface 102 of thespecimen 101 around the second horizontal axis 149, so that a pluralityof specimen locations on the surface of the specimen 101 can be probed.When the light source 110 emits the source light, the optical probes 130receive the source light from the light source 110, and deliver thesource light from the working end onto the surface 102 of the specimen101. The source light will then interact with the surface 102 of thespecimen 101 and generate diffused/reflected light. The optical probes130 then collect from the working end the diffused/reflected lightgenerated from interaction of the source light with the specimen 101,and send the collected diffused/reflected light to the spectrometer 120.The collected diffused/reflected light is used to evaluate the marginstatus of the specimen 101. The camera 145 captures images of thespecimen 101, and the images are used to reconstruct the 3D morphologyof the specimen 101.

FIG. 2C shows schematically a system for evaluating a surgical margin oftumor tissues of a living subject according to certain embodiments ofthe present invention. As shown in FIG. 2C, the system has similarelements with the system as shown in FIG. 2A.

FIGS. 3A and 3B show schematically two views of a system for evaluatinga surgical margin of a specimen of a living subject according to certainembodiments of the present invention. As shown in FIG. 3A, the system200 includes, among other things, a scanner and an optical probe 230.The scanner has a sample holder 246, two motors 241 and 242, twoservomotors 243 and 244, and a camera 245. The motors 241 and 242 andtheir rotation directions are clearly shown on the FIG. 3A. Theservomotor 243 is adapted to move the optical probe up (non-contactmode) and down (in contact mode for measurement). The mobile sampleholder 246 is placed underneath the specimen 201 to hold the specimen201 and to prevent the specimen 201 from sagging during measurement. Thesample holder 246 can move up and down by activating the servomotor 244,and is in contact with the specimen 201 only during the measurementtime. The camera 245, such as a webcam camera, is used in thisprototype. The webcam captures images of the specimen 201 at differentangles, allowing the 3D reconstruction of the specimen 201.

FIG. 4 shows schematically (A) a multi-fiber probe according to certainembodiments of the invention, (B) multi-fibers aligned at the entranceof the spectrometer according to certain embodiments of the invention,and (C) a zoom view of the working end of the probe according to certainembodiments of the invention. As shown in FIG. 4A, the optical probe 330includes a plurality of optical fibers spatially arranged in a fiberarray. In certain embodiments, the optical fibers include a source fiber332 for delivering the source light emitted from the light source to thesurface of the specimen, and a plurality of collection fibers 334 forcollecting the diffused/reflected light generated from the interactionof the source light with the specimen. For the illustration purpose, theoptical probe as shown in FIG. 4 uses the SORS technique and has thedepth resolution of about 2 mm, optimized for breast margin evaluation.

The optical probe 330 as shown in FIG. 4 is an improved version of apreviously developed optical probe (described in U.S. Publication No.2010/0145200 A1). As shown in FIG. 4A, the optical probe 330 uses 36fibers divided into 4 quadrants; each quadrant contains three rings.Each quadrant is similar in concept to the original probe built so thata total of 36 fibers is aligned at the entrance of the spectrometer asshown in FIG. 4B. The source fiber 332, which serves as the source ofthe optical probe 330, is positioned in a center of the fiber array. Thecollection fibers 334, which serve as the detectors of the optical probe330, are positioned in the three rings having a center at the sourcefiber 332 such that each collection fiber 334 is offset from the sourcefiber 332. The main idea of the design of the optical probe 330 is toincrease the number of collection fibers so that a larger area may bemeasured in a single integration, thus decreasing the number ofmeasurements needed to cover the sample surface. FIG. 4C shows thefibers with inline filtering to reject fiber signal according to certainembodiments of the present invention.

FIG. 5 shows the source-detector (S-D) offset for an equivalent S/N ofthe optical probe according to certain embodiments of the presentinvention. The S-D offset represents the offset between the source(source fiber 332) and the detector (collection fibers 334) of eachring. The S-D offset of each ring is carefully calculated so that anequivalent S/N can be obtained, as shown in FIG. 5. For example, in theexemplary embodiment shown in FIG. 4A, the offset of the collectionfibers in the first ring and the source fiber is about 1.57 mm; theoffset of the collection fibers in the second ring and the source fiberis about 2.68 mm; and the offset of the collection fibers in the thirdring and the source fiber is about 3.50 mm, respectively. In certainembodiments, Monte Carlo simulation was used to obtain the offset ofFIG. 5 where the number of photons as a function of the S-D offset isshown. The square dots represent the simulation at different S-D and theblue curve represents the logarithm fitting curve. The trianglesrepresent position of each ring allowing an equivalent number of photonsto reach each ring according to certain embodiments of the presentinvention.

FIG. 6 shows schematically (A) a breast resected specimen according tocertain embodiments of the present invention, and (B) tumor positivemargins and negative margins according to certain embodiments of thepresent invention. As shown in FIG. 6A, the specimen 600 includes anormal tissue 610 and a tumor tissue 620. The outer boundary of thespecimen 600 is the surgical margin 630, and the boundary between thenormal tissue 610 and the tumor tissue 620 is the actual tumor boundary650. A hypothesis tumor boundary 640 is predetermined to have apredetermined distance d inward from the surgical margin 630. In otherwords, the predetermined distance d exists between the surgical margin630 and the hypothesis tumor boundary 640. At different positions of thespecimen 600, the predetermined distance d may be longer or shorter thanthe actual distance between the surgical margin 630 and the actual tumorboundary 650. As shown in FIG. 6B, a positive margin exists when thepredetermined distance d is longer than the actual distance between thesurgical margin and the actual tumor boundary, and a negative marginexists when the predetermined distance d is shorter than the actualdistance between the surgical margin and the actual tumor boundary.

In the previous patent application of U.S. Publication No. 2010/0145200A1, the feasibility of using spatially offset Raman spectroscopy (SORS)to evaluate margin status on intact breast specimens has beendemonstrated. The principle of the SORS method in evaluation of themargin is shown on FIG. 6B. For this technique to become standardoperating room practice, simultaneous or sequential measurements of theentire tissue surface will be required.

FIG. 7 shows the margin status of a phantom sample that mimics anexcised cancer tumor according to one embodiment of the invention. Asshown in FIG. 7, the sphere-shaped phantom sample has four regions thathave different biological components in its margin. The sample ismounted on the scanner and the whole surface is automatically measured.Based on the spectrum recorded at each location, a classification methodbasing on principal component analysis algorithm, automaticallyevaluates if the margin status at that point is either positive ornegative.

As can be seen in FIG. 7, the four regions 711-714, marked in red,correspond to four positive margin locations exactly detected andlocated. The green regions 720 correspond to the safe margin regions.The morphology of the sample is correctly reconstructed. This exampledemonstrates the capacity of the system to perform margin assessment forthe entire sample surface. Highly detailed images of the margin statuscan be obtained and illustrated in a manner that the surgeon can easilyrecognize the exact location of any detected positive margin.

Light based methods have the potential to provide automated, fastdetermination of tumor cells in the tumor bed while the patient is stillin the operating room without removing any tissue for such analysis. Incertain embodiments, an intraoperative device using Raman spectroscopyand fluorescence spectroscopy is presented.

Fluorescence Spectroscopy

Intrinsic fluorescence (or autofluorescence) results from naturalbiological fluorophores such as flavins, porphyrins, collagen, elastin,and nicotinamide adenine dinucleotide (NAD). Autofluorescence spectrahave been shown to differ between normal and neoplastic tissues invarious organ systems possibly due to changes in fluorophoreconcentration or environment with the progression of disease [7].Studies which exploit autofluorescence differences for tissuediscrimination have been carried out for the brain [8-11], bronchus[12], colon [13], cervix [14], bladder [15], esophagus [16], skin [17],breast [18], and arterial wall [19]. Recently, fluorescence spectralimaging was also used to evaluate the status of breast surgical marginswith a classification accuracy 85% sensitivity [20].

Raman Spectroscopy

Raman spectroscopy is a technique that measures energy shifts ofscattered light from a sample. When the sample is irradiated with light,while the majority of the photon will be scattered elastically, a smallfraction is scattered inelastically, resulting in an energy shift fromthe incident light. A sample's Raman spectrum is a biochemical figureprint of its molecular structural. Many biological molecules havedistinguishable spectra, so that the biochemical composition of a tissuecan be determined from its Raman spectrum. One particularly relevantbiochemical change for cancer cells is an increase in the nucleic acidcontent concomitant with increased proliferation and geneticinstability. This change, among others such as changes in glycogen andcollagen, can be detected with Raman spectroscopy [21,22].

As with most of the optical methods, several research groups includingthe PI's mentor have exploited Raman spectroscopy for the diagnosis ofdisease in many organs, including the cervix [23,24], bladder andprostate [25], lung [26], skin [27,28], and GI tract [29-31]. Recentlyour laboratory has reported an in vitro study where Raman spectroscopywas employed to discriminate negative or positive margins of breasttissue samples with 95% sensitivity and 100% specificity [32,33].

Combination of Fluorescence Spectroscopy and Raman Spectroscopy

Although Raman and fluorescence are two distinct phenomena, they occurat the same time when a light is shone on the sample. The Raman andfluorescence signals of a sample can be simultaneously measured usingthe same instrument. In certain embodiments, the Raman and fluorescencesignals may be obtained using non-invasive optical technique thatreveals the biochemical makeup of tissues by measuring the vibrationaleffects of inelastic light scattering on chemical bonds. The Raman andfluorescence signals are sensitive to molecular composition andmicro-environment, and are ideal for in situ clinical diagnosis becauseit is rapid, specific, and non-destructive.

In certain embodiments, evaluation may be performed by: obtaining a rawspectrum from the optical data; generating a fluorescence spectrum (FS)and a Raman spectrum (RS) from the raw spectrum; and determining themargin status according to the FS and the RS.

FIG. 8 shows the measured spectrums of a tissue sample according tocertain embodiments of the present invention, wherein (A) shows a rawspectrum, and (B) shows a fluorescence spectrum (FS) and a Ramanspectrum (RS) obtained from the raw spectrum of (A). As shown in FIG.8A, the raw spectrum contains both Raman and fluorescence information.The fluorescence signal can be precisely approximated using a polynomialfitting (black dot curves under the spectrum in FIG. 8A and top curve onFIG. 8B). The Raman spectrum, which is obtained by subtracting the dataof the raw spectrum from the fluorescence contribution, is shown on thebottom of FIG. 8B.

There have been some reports of Raman methods for STS differentialdiagnosis. For example, Manoharan et al. have identified features inRaman spectra that can be used to diagnosis liposarcoma from normaladipose tissue which can also be used to determine tumor grade [34].Kast et al. have used Raman spectroscopy to differentiaterhabdomyosarcoma, Ewing sarcoma, neuroblastoma, and non-Hodgkin'slymphoma with 100% accuracy [35]. Using exogenous fluorescencespectroscopy, Eward et al. have demonstrated the feasibility ofassessment of large surfaces in the tumor beds in animals [36]. However,the use of potentially toxic exogenous fluorescent dyes makes thisapproach less practical and thus limits its impact.

In certain embodiments, the combination of RS and FS may be used toprovide a complete assessment of the tumor bed after STS excision.Within seconds, the device can localize any tumor residual and report tothe surgeon. The complete removal of the tumor is thus ensured; the needfor a second operation is greatly reduced or eliminated.

In certain embodiments, evaluation may be performed by usingfluorescence imaging with the camera (for example, the camera 145 asshown in FIG. 2B) and the Raman spectrum obtained with the opticalprobe. In this case, the evaluation may be performed by: obtaining a rawspectrum from the optical data; generating a Raman spectrum (RS) fromthe raw spectrum; generating a fluorescence image (FI) from the imagesacquired by the camera; and determining the margin status according tothe FI and the RS.

One aspect of the present invention relates the use of emerging provenmethods to a new field of clinical challenge. No other group, to thebest of applicant's knowledge, has applied Raman spectroscopy andintrinsic fluorescence spectroscopy to assess the tumor bed after softtissue sarcoma excision. All the above mentioned works focus on using RSin differentiate several STS tissue types or using exogenous FS inassessing the tumor bed, with prior intravenous injection of fluorescentdyes. The combination of RS and FS proposed here could offer significantimprovements over conventional approaches.

In addition, a further aspect of the invention relates to softwareimplementation (i.e., computer executable instruction codes) stored in anon-transitory computer-readable medium which, when executed by aprocessor, cause a system to perform a method for evaluating a surgicalmargin of a specimen of a living subject. In certain embodiments, thesoftware may be written in Labview and Matlab to perform the followingtasks:

(1) Reconstructing the 3D morphology of the sample;

(2) Controlling of the scanner so that the entire sample surface can beautomatically measured;

(3) Evaluating the margin status based on the presence of a cancersignal in spectra recorded at each location; and

(4) Displaying the margin status in 3D with morphological orientations.

In one embodiment, a system to perform the method includes a pluralityof parts, which are listed in Table 1.

TABLE 1 parts used to construct the system Number Part Quantity Utility1 Motor 2 Move the sample and optical probes 2 Servomotor 4 Move theprobes and sample holder 3 Electronic 2 Control the motors andservomotors Board 4 Camera ≧1 Take images of the specimen 5 Optical ≧1Measure optical signal from the probes specimen 6 Spectrometer ≧1Analyze the optical signal and Detector 7 PC and 1 Control measurementsand analyze Software data

Referring to FIG. 9, a flowchart 900 of a method for evaluating asurgical margin of a specimen of a living subject is shown according toone embodiment of the invention. The method includes the followingsteps: at step 910, images of the specimen are acquired. At step 920, a3D morphological surface of the specimen is calculated from the acquiredspecimen images, and the 3D morphological surface of the specimen isdisplayed. At step 930, specimen locations are obtained from the 3Dmorphological surface to cover the entire surface of the specimen.

At step 940, optical data is acquired at each specimen location. In oneembodiment, the acquiring step includes providing a source light;delivering the source light at each specimen location onto a surface ofthe specimen; and collecting diffused/reflected light generated frominteraction of the source light with the specimen at the specimenlocation. In addition, the acquiring step is performed with at least oneoptical probe. In one embodiment, each optical probe includes aplurality of optical fibers spatially arranged in a fiber array. Incertain embodiments, for each optical probe, the optical fibers includea source fiber for delivering the source light emitted from the lightsource to the surface of the specimen, and a plurality of collectionfibers for collecting the diffused/reflected light generated from theinteraction of the source light with the specimen. In certainembodiments, the source fiber is positioned in a center of the fiberarray, and the plurality of collection fibers is positioned in one ormore rings having a center at the source fiber such that each collectionfiber is offset from the source fiber, as shown in FIG. 4.

Then, at step 950, a margin status of the specimen at each specimenlocation is evaluated to either positive or negative based on theacquired optical data. In certain embodiments, the evaluating step maybe performed by: obtaining a raw spectrum, as shown in FIG. 8A, from theoptical data; generating a fluorescence spectrum (FS) and a Ramanspectrum (RS), as shown in FIG. 8B, from the raw spectrum; anddetermining the margin status according to the FS and the RS.

Finally, at step 960, the margin status of the specimen on the 3Dmorphological surface of the specimen with morphological orientations isdisplayed.

Additionally, the method may further include cooperatively moving thespecimen and the at least one optical probe to acquire the optical dataat all of the specimen locations to cover the entire surface of thespecimen.

In certain embodiments, the method may further include: mounting thespecimen in a scanner; and calibrating a position of the specimen in thescanner. In certain embodiments, the scanner includes: a sample holderfor holding the specimen; a first motor for rotating the specimen alonga first horizontal axis; and a second motor for moving the at least oneoptical probe along a surface of the specimen.

In certain embodiments, the steps 910-960 are all automaticallyperformed by the software. However, manual intervention is needed at thebeginning, in which the sample is mounted in the sample holder and theposition of the sample is calibrated.

One aspect of the present invention relates to a non-transitorycomputer-readable medium storing computer executable instructions which,when executed by a processor, cause a system to perform a method forevaluating a surgical margin of tumor tissues of a living subject. Incertain embodiments, the method performed by the system may be themethod as disclosed in the flowchart of FIG. 9.

Example One

In this example, in vitro study is performed. Preliminary Raman andfluorescence spectra have been acquired to (1) demonstrate the abilityof Raman and fluorescence spectroscopy in differentiating STS and normaltissues and (2) confirm the feasibility of measuring Raman andfluorescence signal of tumor beds in the operating room.

Under VU IRB approval (#120813), tumor and control samples from 20patients have been acquired from the VU Cooperative Human TissueNetwork. Raman spectra of the samples were collected using a portablefiber optic system, yielding 44 spectra from tumors and 33 spectra fromcontrol muscles.

FIG. 10A shows schematically average Raman spectra of control muscle andSTS samples according to certain embodiments of the present invention.Multivariate statistical analysis was used to classify spectra intotumor and control groups with 100% sensitivity and 100% specificity, asshown in Table 2.

TABLE 2 Classification matrix for in vitro specimens Muscle TumorHistopathology Tumor 0 44 Sensitivity: 100% Muscle 32 0 Specificity:100%

FIG. 10B shows schematically mean Raman spectra and associatedbiochemical peaks according to certain embodiments of the presentinvention. As shown in FIG. 10B, statistical significance is indicatedby asterisks (*: p<0.01; **: p<0.5).

The in vitro data also suggest that FS can be used to differentiatemuscle and STS. FIG. 11 shows (a) fluorescence spectra of muscle andSTS, (b) an image of the surface of a samples and the boundary of STSand normal muscle, and (c) fluorescence intensity (mean±standarddeviation) of the two regions according to certain embodiments of thepresent invention. As shown in FIG. 11(a), the fluorescent spectrum ofhealthy muscle and STS sample, measured under the same settings. It canbe seen that muscle exhibits a much stronger fluorescence signal. Asshown in FIG. 11(b), a tissue sample consists of healthy muscle on theright side, and STS on the left side, is used to demonstrated thefeasibility of using fluorescence imaging to differentiate STS andmuscle. A two dimensional map (˜5 mm×3 mm) of the sample is measured andat each point on the surface, a spectrum is acquired. The data isprocessed and fluorescence information is extracted. Based on thefluorescence intensity, a threshold has been chosen to classify thesample into two regions, corresponding to tumor (red) and muscle(green). As shown in FIG. 11(c), the fluorescence intensity of those tworegions is statistically different (p<0.0001). The border of STS andmuscle is demarcated by the red curve in FIG. 11(b) and is confirmedhistologically.

FIG. 12 shows the probability of individual samples being either normalmuscle or tumor muscle according to certain embodiments of the presentinvention. These results suggest the potential of Raman and fluorescencespectroscopy in differentiating soft tissue sarcomas and healthy musclewith 100% accuracy.

Example Two

In this example, pilot clinical study is performed. The initial workdemonstrated the feasibility of using Raman and fluorescencespectroscopy to differentiate STS and muscle on intact specimens in alaboratory setting. Studies are currently underway using the sameapproach in a clinical setting, and results are equally promising. Ramanspectra were recorded for 3-10 seconds from multiple sites in the tumorbed of 19 patients undergoing STS excision at the VU Medical Center.

FIG. 13 shows schematically Raman spectra of in vivo tissues accordingto certain embodiments of the present invention. As shown in FIG. 13,spectrum from 1 to 8 is the Raman spectrum of healthy tissues, as data 1represents synovium; data 2 represents tendon; data 3 represents skin;data 4 represents nerve; data 5 represents bone marrow; data 6represents fat; data 7 represents bone; and data 8 represents muscle.The Raman spectra of STS tumors from selected 4 patients are marked asp1-4 and are histologically confirmed to be: p1—round cell sarcoma;p2—malignant peripheral nerve sheath tumor; p3—undifferentiatedpleomorphic sarcoma; and p4—undetermined STS.

Based on direct visualization, Raman spectra of STS (p1-p4) looksimilar, but they are different from the healthy ones (spectrum from 1to 8). The gray bands highlight the spectral regions subject to the mostdramatic differences. These include the difference of the 1006 cm⁻¹ peakgenerally attributed to phenylalanine; a decreasing of the 1265 and 1303cm⁻¹ peaks, which tends to indicate a change in protein content; and theincreasing width of the amide I peak around 1656 cm⁻¹. These significantspectral differences indicate the feasibility of RS in differentiatingSTS from normal tissues. A total of 396 spectra acquired from those 19patients (in which 304 from healthy tissues and 86 from tumor) are usedas an input of a support vector machine (SVM) classification algorithm(C=10 and gamma=0.001). All 304 spectra from healthy tissues and 92 fromSTS were predicted to have the right diagnosis with excellentsensitivity (94.6%) and specificity (98.4%), as shown in Table 3.

TABLE 3 Classification matrix for in vivo specimens Normal TumorHistopathology Tumor 5 87 Sensitivity: 94.6% Normal 301 3 Specificity:98.4%

The use of fluorescence spectroscopy in discriminating STS tumor andnormal muscle is also evaluated. FIG. 14 shows fluorescence intensity(mean±standard deviation) of muscle and tumor from 19 patients accordingto certain embodiments of the present invention. As shown in FIG. 14,the fluorescence intensity of STS is found to be statistically smallerthan that of the healthy muscle in all patients. These results indicatethat fluorescence has the potential to be an excellent optical tool tolocate STS in muscle tissues.

The preliminary results described above show the ability of Raman andfluorescence spectroscopy in differentiating STS from normal tissueswith excellent sensitivity (94.6%) and specificity (98.4%). The pilotstudy also demonstrates the feasibility of acquiring good quality Ramanand fluorescence data in clinical settings.

In sum, the present invention, among other things, recites methods andsystems for evaluating surgical margins of tumor tissues of a livingsubject. In certain embodiments, Raman and fluorescence spectroscopy canbe used to effectively discriminate between normal and tumor tissueduring soft tissue sarcoma margin assessment.

The foregoing description of the exemplary embodiments of the inventionhas been presented only for the purposes of illustration and descriptionand is not intended to be exhaustive or to limit the invention to theprecise forms disclosed. Many modifications and variations are possiblein light of the above teaching.

The embodiments were chosen and described in order to explain theprinciples of the invention and their practical application so as toenable others skilled in the art to utilize the invention and variousembodiments and with various modifications as are suited to theparticular use contemplated. Alternative embodiments will becomeapparent to those skilled in the art to which the present inventionpertains without departing from its spirit and scope. Accordingly, thescope of the present invention is defined by the appended claims ratherthan the foregoing description and the exemplary embodiments describedtherein.

REFERENCE LIST

-   [1] American Cancer Society. Cancer Facts & Figures 2012. Atlanta:    American Cancer Society,    <http://www.cancer.org/acs/groups/content/@epidemiologysurveilance/document    s/document/acspc-031941.pdf (2012).-   [2] Atean, I. et al. Prognostic factors of extremity soft tissue    sarcoma in adults. A single institutional analysis.    Cancer/Radiothérapie 16, 661-666,    doi:http://dx.doi.org/10.1016/j.canrad.2012.05.021 (2012).-   [3] Blakely, M. L. et al. The impact of margin of resection on    outcome in pediatric nonrhabdomyosarcoma soft tissue sarcoma.    Journal of Pediatric Surgery 34, 672-675 (1999).-   [4] Gronchi, A. et al. Status of Surgical Margins and Prognosis in    Adult Soft Tissue Sarcomas of the Extremities: A Series of Patients    Treated at a Single Institution. Journal of Clinical Oncology 23,    96-104, doi:10.1200/jco.2005.04.160 (2005).-   [5] Grimer, R., Judson, I., Peake, D. & Seddon, B. Guidelines for    the management of soft tissue sarcomas. Sarcoma 506182, 31 (2010).-   [6] Gould, S. W. et al. Resection of soft tissue sarcomas with    intra-operative magnetic resonance guidance. J Magn Reson Imaging    15, 114-119 (2002).-   [7] Lakowicz, J. R. Principles of Fluorescence Spectroscopy. (Plenum    Press, 1983).-   [8] Lin, W. C., Toms, S. A., Motamedi, M., Jansen, E. D. &    Mahadevan-Jansen, A. Brain tumor demarcation using optical    spectroscopy; an in vitro study. J Biomed Opt 5, 214-220. (2000).-   [9] Lin, W. C., Toms, S. A., Johnson, M., Jansen, E. D. &    Mahadevan-Jansen, A. In vivo brain tumor demarcation using optical    spectroscopy. Photochem Photobiol 73, 396-402. (2001).-   [10] Chung, Y. G., Schwartz, J. A., Gardner, C. M., Sawaya, R. E. &    Jacques, S. L. Diagnostic potential of laser-induced    autofluorescence emission in brain tissue. J Korean Med Sci 12,    135-142. (1997).-   [11] Bottiroli, G. et al. Brain tissue autofluorescence: an aid for    intraoperative delineation of tumor resection margins. Cancer Detect    Prev 22, 330-339 (1998).-   [12] Zellweger, M. et al. In vivo autofluorescence spectroscopy of    human bronchial tissue to optimize the detection and imaging of    early cancers. J Biomed Opt 6, 41-51. (2001).-   [13] Richards-Kortum, R. et al. Spectroscopic diagnosis of colonic    dysplasia. Photochem Photobiol 53, 777-786. (1991).-   [14] Ramanujam, N. et al. Cervical precancer detection using a    multivariate statistical algorithm based on laser-induced    fluorescence spectra at multiple excitation wavelengths. Photochem    Photobiol 64, 720-735. (1996).-   [15] D'Hallewin, M. A., Baert, L. & Vanherzeele, H. Fluorescence    imaging of bladder cancer. Acta Urol Belg 62, 49-52. (1994).-   [16] Panjehpour, M. et al. Spectroscopic diagnosis of esophageal    cancer: new classification model, improved measurement system.    Gastrointest Endosc 41, 577-581 (1995).-   [17] Chwirot, B. W., Chwirot, S., Redzinski, J. & Michniewicz, Z.    Detection of melanomas by digital imaging of spectrally resolved    ultraviolet light-induced autofluorescence of human skin. Eur J    Cancer 34, 1730-1734. (1998).-   [18] Gupta, P. K., Majumder, S. K. & Uppal, A. Breast cancer    diagnosis using N2 laser excited autofluorescence spectroscopy.    Lasers Surg Med 21, 417-422 (1997).-   [19] Warren, S. et al. Combined ultrasound and fluorescence    spectroscopy for physico-chemical imaging of atherosclerosis. IEEE    Trans Biomed Eng 42, 121-132. (1995).-   [20] Keller, M. D. et al. BSuB6 (Optical Society of America).-   [21] Mahadevan-Jansen, A. in Raman Spectroscopy: From Benchtop to    Bedside, Biomedical Photonics Handbook, (ed 30:1-30:27 T. Vo-Dinh,    CRC Press, Washington D.C., 2003.).-   [22] Mahadevan-Jansen, A. & Richards-Kortum, R. R. Raman    spectroscopy for the detection of cancers and precancers. Journal of    biomedical optics 1, 31-70, doi:10.1117/12.227815 (1996).-   [23] Mahadevan-Jansen, A. et al. Near-infrared Raman spectroscopy    for in vitro detection of cervical precancers. Photochem Photobiol    68, 123-132 (1998).-   [24] Mahadevan-Jansen, A., Mitchell, M. F., Ramanujam, N.,    Utzinger, U. & Richards-Kortum, R. Development of a fiber optic    probe to measure NIR Raman spectra of cervical tissue in vivo.    Photochem Photobiol 68, 427-431 (1998).-   [25] Crow, P. et al. Assessment of fiberoptic near-infrared raman    spectroscopy for diagnosis of bladder and prostate cancer. Urology    65, 1126-1130, doi:10.1016/j.urology.2004.12.058 (2005).-   [26] Huang, Z. et al. Near-infrared Raman spectroscopy for optical    diagnosis of lung cancer. International journal of cancer. Journal    international du cancer 107, 1047-1052, doi:10.1002/ijc.11500    (2003).-   [27] Lieber, C. A., Majumder, S. K., Ellis, D. L., Billheimer, D. D.    & Mahadevan-Jansen, A. In vivo nonmelanoma skin cancer diagnosis    using Raman microspectroscopy. Lasers Surg Med 40, 461-467 (2008).-   [28] Sigurdsson, S. et al. Detection of skin cancer by    classification of Raman spectra. IEEE transactions on bio-medical    engineering 51, 1784-1793, doi:10.1109/TBME.2004.831538 (2004).-   [29] Shetty, G., Kendall, C., Shepherd, N., Stone, N. & Barr, H.    Raman spectroscopy: elucidation of biochemical changes in    carcinogenesis of oesophagus. British journal of cancer 94,    1460-1464, doi:10.1038/sj.bjc.6603102 (2006).-   [30] Shim, M. G., Song, L. M., Marcon, N. E. & Wilson, B. C. In vivo    near-infrared Raman spectroscopy: demonstration of feasibility    during clinical gastrointestinal endoscopy. Photochem Photobiol 72,    146-150 (2000).-   [31] Molckovsky, A., Song, L. M., Shim, M. G., Marcon, N. E. &    Wilson, B. C. Diagnostic potential of near-infrared Raman    spectroscopy in the colon: differentiating adenomatous from    hyperplastic polyps. Gastrointestinal endoscopy 57, 396-402,    doi:10.1067/mge.2003.105 (2003).-   [32] Keller, M. D. et al. Development of a spatially offset Raman    spectroscopy probe for breast tumor surgical margin evaluation.    Journal of biomedical optics 16, 077006, doi:10.1117/1.3600708    (2011).-   [33] Mahadevan-Jansen A et al. Looking Below the Surface of Breast    Tissue during Surgery. Spectroscopy (2011).-   [34] Manoharan, R. et al. in SPIE (Europe). 128-132 (Bellingham).-   [35] Kast, R. et al. Differentiation of small round blue cell tumors    using Raman spectroscopy. Journal of Pediatric Surgery 45,    1110-1114, doi:http://dx.doi.org/10.1016/j.jpedsurg.2010.02.072    (2010).-   [36] Eward, W. et al. A Novel Imaging System Permits Real-time in    Vivo Tumor Bed Assessment After Resection of Naturally Occurring    Sarcomas in Dogs. Clin Orthop Relat Res, 1-9,    doi:10.1007/s11999-012-2560-8 (2012).

What is claimed is:
 1. A method of evaluating a surgical margin of tumortissues of a living subject, comprising: (a) acquiring images of aspecimen of the tumor tissues; (b) calculating a three-dimensional (3D)morphological surface of the specimen from the acquired images anddisplaying the 3D morphological surface; (c) obtaining, from the 3Dmorphological surface, a plurality of specimen locations to cover asurface of the specimen; (d) acquiring optical data at each specimenlocation; (e) evaluating a margin status of the specimen at eachspecimen location to either positive or negative based on the acquiredoptical data; and (f) displaying the margin status of the specimen onthe 3D morphological surface of the specimen with morphologicalorientations.
 2. The method of claim 1, wherein the step of acquiringthe optical data comprises: providing a source light; delivering thesource light at each specimen location onto a surface of the specimen;and collecting diffused/reflected light generated from interaction ofthe source light with the specimen at the specimen location.
 3. Themethod of claim 2, wherein the step of acquiring the optical data isperformed with at least one optical probe or at least one detector. 4.The method of claim 3, wherein each optical probe comprises a pluralityof optical fibers spatially arranged in a fiber array.
 5. The method ofclaim 4, wherein for each optical probe, the optical fibers comprise asource fiber for delivering the source optical light emitted from thelight source to the surface of the specimen, and a plurality ofcollection optical fibers for collecting the diffused/reflected lightgenerated from the interaction of the source light with the specimen. 6.The method of claim 5, wherein the source optical fiber is positioned ina center of the fiber array, and the plurality of collection opticalfibers is positioned in one or more rings having a center at the sourceoptical fiber such that each collection fiber is offset from the sourceoptical fiber.
 7. The method of claim 3, further comprising:cooperatively moving the specimen and the at least one optical probe toacquire the optical data at all of the specimen locations.
 8. The methodof claim 3, further comprising: mounting the specimen in a scanner; andcalibrating a position of the specimen in the scanner.
 9. The method ofclaim 8, wherein the scanner comprises: a first motor for rotating thespecimen along a first horizontal axis; and a second motor for movingthe at least one optical probe along a surface of the specimen.
 10. Themethod of claim 9, wherein the scanner further comprises a camera foracquiring images of the specimen so as to reconstruct athree-dimensional (3D) morphological surface of the specimen.
 11. Themethod of claim 10, wherein the evaluating step comprises: obtaining araw spectrum from the optical data; generating a Raman spectrum (RS)from the raw spectrum; and determining the margin status according tothe RS.
 12. The method of claim 10, wherein the evaluating stepcomprises: generating a fluorescence signal from the images acquired bythe camera; and determining the margin status according to thefluorescence signal.
 13. The method of claim 1, wherein the evaluatingstep comprises: obtaining a raw spectrum from the optical data;generating a fluorescence spectrum (FS) and a Raman spectrum (RS) fromthe raw spectrum; and determining the margin status according to the FSand the RS.
 14. A non-transitory computer-readable medium storingcomputer executable instructions which, when executed by a processor,cause a system to perform a method for evaluating a surgical margin oftumor tissues of a living subject, the method comprising: (a) acquiringimages of a specimen of the tumor tissues; (b) calculating athree-dimensional (3D) morphological surface of the specimen from theacquired images and displaying the 3D morphological surface; (c)obtaining, from the 3D morphological surface, a plurality of specimenlocations to cover a surface of the specimen; (d) acquiring optical dataat each specimen location; (e) evaluating a margin status of thespecimen at each specimen location to either positive or negative basedon the acquired optical data; and (f) displaying the margin status ofthe specimen on the 3D morphological surface of the specimen withmorphological orientations.
 15. The non-transitory computer-readablemedium of claim 14, wherein the step of acquiring the optical datacomprises: providing a source light; delivering the source light at eachspecimen location onto the surface of the specimen; collectingdiffused/reflected light generated from interaction of the source lightwith the specimen at the specimen location; and evaluate the marginstatus of the specimen from the collected diffused/reflected light ateach specimen location of the surface of the specimen.
 16. Thenon-transitory computer-readable medium of claim 15, wherein the step ofacquiring the optical data is performed with at least one optical probeor at least one detector.
 17. The non-transitory computer-readablemedium of claim 16, wherein the method further comprises cooperativelymoving the specimen and the at least one optical probe to acquire theoptical data at all of the specimen locations.
 18. The non-transitorycomputer-readable medium of claim 14, wherein the evaluating stepcomprises: obtaining a raw spectrum from the optical data; generating afluorescence spectrum (FS) and a Raman spectrum (RS) from the rawspectrum; and determining the margin status according to the FS and theRS.
 19. The non-transitory computer-readable medium of claim 14, whereinthe method further comprises: mounting the specimen in a scanner; andcalibrating a position of the specimen in the scanner.
 20. Thenon-transitory computer-readable medium of claim 19, wherein the scannercomprises a camera for acquiring images of the specimen so as toreconstruct a three-dimensional (3D) morphological surface of thespecimen.
 21. The non-transitory computer-readable medium of claim 20,wherein the evaluating step comprises: obtaining a raw spectrum from theoptical data; generating a Raman spectrum (RS) from the raw spectrum;and determining the margin status according to the RS.
 22. Thenon-transitory computer-readable medium of claim 20, wherein theevaluating step comprises: generating a fluorescence signal from theimages acquired by the camera; and determining the margin statusaccording to the fluorescence signal.